Dengue virus (DENV) belongs to the Flaviviridae family of enveloped, positive-strand RNA viruses, and is transmitted by Aedes mosquitoes. DENV infection is the most important arthropod-borne viral disease, with about 390 million infections every year, that can result in dengue fever (DF), and in 1-5% of cases in dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), characterized by vascular leak leading to hypotensive shock (World Health Organization (WHO) Press 2009, WHO/HTM/NTD/DEN/2009. 1 ed.; Bhatt S et al., Nature 2013, 496(7446):504-507). Over 2 million cases of severe dengue disease and over 20,000 deaths are estimated to occur each year (Gubler D J, The American journal of tropical medicine and hygiene 2012, 86(5):743-744).
There are four main DENV serotypes (designated DENV1-4) that are 67-75% identical at the amino acid level. The viral RNA genome is translated as a single polyprotein that is cleaved by viral and host proteases into three structural proteins (capsid (C), premembrane (prM), and envelope (E)) and seven non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5). Complete nucleotide sequences of the reference genomes of the 4 dengue virus serotypes can be accessed from the Genbank database under accession numbers NC_001477.1, NC_001474.2, NC_001475.2 and NC_002640.1 respectively. During infection, the E protein interacts with cellular receptors and viral uptake occurs via receptor-mediated endocytosis (Heinz F X et al., Archives of virology Supplementum 1994, 9:339-348; Mukhopadhyay S et al., Nat Rev Microbiol 2005, 3(1):13-22). The E protein is structurally conserved among flaviviruses and consists of three domains (EDI, EDII, and EDIII) (Rey F A et al., Nature 1995, 375(6529):291-298). Notably, the EDIII domain induces the most potent neutralizing and serotype-specific antibodies (Beltramello M et al., Cell host & microbe 2010, 8(3):271-283; Shrestha B et al., PLoS Pathog 2010, 6(4):e1000823; Sukupolvi-Petty S et al., J Virol 2010, 84(18):9227-9239; Wahala W M et al., PLoS Pathog 2010, 6(3):e1000821; de Alwis R et al, PLoS neglected tropical diseases 2011, 5(6):e1188; Yauch L E et al., Advances in virus research 2014, 88:315-372).
A single minimal tetravalent DENV antigen composed of the four envelope domain III (EDIII) from the four DENV serotypes fused to the ectodomain of the membrane protein (ectoM) has been previously described (Brandler et al., PLoS 2007, 1(3), e96; Brandler et al., Vaccine, 2010, 28, 6730-6739). When expressed by a replicating viral vector derived from live-attenuated MV vaccine, this antigen induced neutralizing antibodies against the four serotypes of dengue virus (Brandler et al., Vaccine, 2010, 28, 6730-6739). However, evaluated in a non-human primate model of DENV infection, a recombinant MV vector expressing the tetravalent EDIII-ectoM antigen provided only partial protection. This observation indicated that an additional DENV antigen was missing to provide full protection.
The NS proteins are involved in viral replication and assembly and are usually not incorporated in mature viral particles. Strikingly, while a primary infection by one DENV serotype induces a lasting protective immunity against reinfection by the same serotype, not only it does not protect against infection with other serotypes but it increases also the risk to develop a more severe disease upon secondary infection, a phenomenon attributed to non-neutralizing or sub-neutralizing antibodies and called Antibody-Dependent Enhancement (ADE) (Halstead S B et al. Nature 1977, 265(5596):739-741; Dejnirattisai W et al. Science 2010, 328(5979):745-748). In support of the ADE hypothesis, it was proposed that the low levels of serotype cross-reactive antibodies produced following a primary infection, can enhance the secondary infections through the formation of DENV-antibody complexes that bind to the Fcγ receptors (FcγR) on myeloid cells. This process leads to higher viral load and higher production of inflammatory mediators responsible of vascular permeability (Halstead S B et al. Nature 1977, 265(5596):739-741; Morens D M et al., Clinical infectious diseases: an official publication of the Infectious Diseases Society of America 1994, 19(3):500-512; Halstead S B et al., Advances in virus research 2003, 60:421-467).
Mechanistically, ADE was shown to be critically dependent on the activation of FcγRIIa receptor expressed by monocytes, macrophages and dendritic cells (DCs) which produce high amounts of cytokines (TNF-α and IL-6) and chemokines (MIP-1a) upon stimulation (Wong K L et al., PLoS ONE 2012, 7(5):e36435; Boonnak K et al., J Immunol 2013, 190(11):5659-5665; Guilliams M et al., Nat Rev Immunol 2014, 14(2):94-108). The increased viral loads in ADE were also shown to result from the binding of immune complexes (between the virus and sub-neutralizing antibodies) to the leukocyte Ig-like receptor B1 (LILR-B1) on human primary monocytes, leading to the inhibition of the early antiviral responses mediated by the activated FcγRIIa (Chan K R et al., Proc Natl Acad Sci USA 2014, 111 (7):2722-2727).
Thus, depending on the nature of the DENV-specific antibody response, the adaptive immunity can induce either protection against infection or enhancement of infection and disease progression.
Like B cells, a pathogenic role of virus-specific T cells during secondary infection was also suggested. The hypothesis, called “original antigenic sin” postulated that, after a primary infection, cross-reactive memory T cells, with low avidity for the serotype of the secondary infection, dominate and mask the specific T cell response, leading to a less efficient killing of infected target cells (Mongkolsapaya J et al. Nat Med 2003, 9(7):921-927). These cross-reactive CD8+ T cells, stimulated upon a secondary infection with a different serotype, displayed also quantitative and qualitative differences in their response to the cross-reactive epitope or the altered peptide ligand (Bashyam H S et al., J Immunol 2006, 176(5):2817-2824).
However, in spite of these studies, the direct demonstration of a pathogenic role of DENV-specific T cells is still missing, and recent reports did not support a causative role for cross-reactive CD8+ T cells in the pathogenesis of dengue hemorrhagic fever during secondary infections. Indeed, a study in adults experiencing a secondary infection did not reveal any correlation between the magnitude and specificity of T-cell responses and clinical disease grade (Simmons C P et al., J Virol 2005, 79(9):5665-5675), and an important protective role for CD8+ T cells during primary DENV infection was also identified in a mouse model (Yauch L E et al. J Immunol 2009, 182(8):4865-4873). More strikingly, a detailed analysis of HLA-restricted T-cell responses in donors from hyperendemic area even reinforces the protective role of CD8+ T cells during DENV infection (Weiskopf D et al., Proc Natl Acad Sci USA 2013, 110(22):E2046-2053). It appears that, whereas serotype-specific responses are a hallmark of primary infection, there is a shift towards a response against conserved epitopes following secondary infection, without any difference in the avidity or functionality in CD8+ T cells among serotype-specific or conserved responses. In addition, a significant correlation was established between a weak T-cell response and disease susceptibility (Weiskopf D et al., Proc Natl Acad Sci USA 2013, 110(22):E2046-2053). Collectively, these studies highlight the beneficial effect of CD8+ T cells against disease progression in dengue virus infection.